Chromatin remodeling protein as a marker expressed by stromal progenitor cells

ABSTRACT

The present invention provides a chromatin remodeling protein, designated ChroM as well as alternatively spliced variants, which serves as markers for stromal precursor cells and osteogenic and muscle progenitor cells. This ChroM marker was found to be present in proliferating progenitor cells but is either absent or not predominantly present in resting cells. Antibodies directed against this ChroM marker can be used to isolate stromal progenitor cells that can differentiate into osteogenic and muscle cells. Subpopulations of human osteogenic progenitor cells can further be separated using additional markers. Due to the discovery that osteogenic progenitor sarcoma cells can be distinguished from other cells by the presence of ChroM in the nuclei or based on genetic alteration on the DNA level, a method for identifying osteosarcoma cells in a tissue sample and a method for evaluating the effectiveness of a treatment for osteogenic progenitor sarcoma are also provided.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to proteins involved with genome integrityand chromatin-remodeling complexes and their encoding DNA. The presentinvention also relates to antibodies against a chromatin remodelingprotein which can be used to separate stromal mesenchymal includingosteogenic and muscle progenitor cells from a cell mixture, to anisolated population of enriched cells, and to methods of using thechromatin remodeling protein and the isolated population of enrichedcells.

2. Description of the Related Art

Transcriptional regulation of cell differentiation is a complex systemand the multiple factors that play a role in the process of skeletal(bone and muscle) formation from mesenchymal precursors are not wellsorted. Steroid hormones such as estrogen, androgen, glucocorticoids andvitamin D are known to play a major role in regulating physiologicalprocesses of the skeleton (Manolagas et al., 1999 and Spelsberg et al.,1999). Most important is their role relating to the ability of thehormone-bound nuclear receptors (NR) to change the expression of targetgenes in a cell- and promoter-dependent manner.

The transcriptional activity of NR depends on co-activators andco-repressors which regulate transcription by remodeling chromatin or byfacilitating the recruitment of the basal transcriptional machinery.Co-activators are often part of multiprotein complexes which are notspecific and which mediate the activity of other nuclear receptors(NRs). Surprisingly, different tissues respond in a selective manner tothese hormones. Recent studies revealed that the activity ofco-activators may contribute to the receptor, promoter and cellspecificity of NR action (Jenkins et al., 2001 and Bourachot et al.,1999). The coordination of complex and dynamic networks in transcriptionregulation by steroid hormones is linked to other signaling pathways. Abetter understanding of the molecular mechanism underlyingligand-dependent transcriptional activation by nuclear receptors isbelieved to be through accessories proteins, co-activators andco-repressors. The co-activator proteins in complex with nuclearreceptors act to remodel chromatin within the promoter region and torecruit the transcriptional machinery to the promoter in order toinitiate transcription (Dilworth et al., 2000). The regulation of geneexpression is based on transcription from chromatin templates. Chromatinremodeling occurs by the action of enzymes, which require ATP-dependentchromatin remodeling in proteins that are SNF-like, and in HAT enzyme orprotein that possesses histone-binding domains, like the bromodomain,and contributes to the regulation of transcription by influencingactivator-dependent recruitment (Fry et al., 2001). The specificactivities of these proteins are still being explored and have becomethe focus of many research laboratories.

Citation of any document herein is not intended as an admission thatsuch document is pertinent prior art, or considered material to thepatentability of any claim of the present application. Any statement asto content or a date of any document is based on the informationavailable to applicant at the time of filing and does not constitute anadmission as to the correctness of such a statement.

SUMMARY OF THE INVENTION

The present invention is directed to a chromatin remodeling proteindesignated ChroMi, and fragments and variants thereof having theactivity and properties of the chromatin remodeling protein, such asATPase activity, DNA binding, LXXLL motif (NR box) for interaction withnuclear receptors, and GXXXG motif important in interactions withtransmembrane (TM) proteins. The variants may be those having at least95% sequence identity to the amino acid sequence of SEQ ID NO:2 or whichare naturally occurring alternative splice forms.

The present invention also provides a nucleic acid molecule thatcontains the nucleotide sequence encoding ChroM1, or a fragment orvariant thereof, and an antisense oligonucleotide that inhibits theproduction of the chromatin remodeling protein. The antisenseoligonucleotide can be used in a method for treating osteosarcoma.Further provided is an isolated fragment of human genomic DNA whichencodes ChroM1.

Another aspect of the present invention is a molecule having the antigenbinding portion of an antibody specific for the chromatin remodelingprotein of the present invention. This molecule can be used in a methodfor separating a cell population containing human stromal progenitorcells that differentiate into osteogenic and muscle cells from a cellmixture derived from human bone marrow by selectively binding themolecule to ChroM on human stromal progenitor cells. The antibody-boundcells are then separated from the cell mixture in to selective ex vivoculture conditions.

The method can be extended to further separate a subpopulation of humanosteogenic progenitor cells through the use of additional markerspresent on the cells of the subpopulation. The present invention thusalso provides an isolated cell population enriched for human osteogenicprogenitor cells and a composition comprising a physiologicallyacceptable medium and the isolated cell population enriched for humanosteogenic progenitor cells. A further aspect of the present inventionrelates to a method of treating bone or muscle tissue damage and amethod of generating bone or muscle tissue.

Still further aspects of the present invention are directed to a methodof screening for and identifying an enhancer or inhibitor compound thataffect expression of the ChroM1 chromatin remodeling protein, to amethod of identifying AT-rich promoter regions of genes involved inmodulation of osteoblast differentiation and capable of binding to theDNA binding domain of ChroM1, and to a method of screening for andidentifying a compound which stimulates differentiation-of osteogenicprogenitor cells.

Due to the discovery that osteogenic progenitor sarcoma cells can bedistinguished from normal osteoblasts by the presence of ChroM1 in thenuclei, the present invention further provides a method for identifyingosteosarcoma cells in a tissue sample and a method for evaluating theeffectiveness of a treatment, such as chemotherapy to specifically blockthe ChroM1 binding to chromatin and therefore block DNA binding incomplex formation, for osteogenic sarcoma through the use of themolecule according to the present invention which has an antigen bindingportion of an anti-ChroM antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show schematic illustrations of the genomic organizationof exons of ChroM1 by size and number (FIG. 1A). The functional proteinmotifs of ChroM1 are presented in FIG. 1B.

FIGS. 2 show the prediction for phosphorylation on amino acid residuesserine and tyrosine. Immunoprecipitation (Ip) was performed withanti-ChroM antibody and analyzed by Western blot withanti-phosphotyrosine and anti-phosphoserine in the presence (+) orabsence (−) of phosphatase inhibitors (Ph-I).

FIG. 3 shows a graph of the level of expression for ChroM1^(VE+) mRNAcompared between cells derived from cultured trabecular bone (TBC) andthose derived from marrow stromal cultured (MSC). The resultsdemonstrate the distribution of ChroM1^(VE+) cells from differentdonors. The MSC and TBC cells are shown below the graph under the MSCand TBC labels.

FIG. 4 shows a graph of a RT-PCR analysis of the differential expressionof ChroM1 in osteogenic/non-osteogenic clonal MSC population.

FIG. 5 shows in vivo staining of osteoprogenitor cells in tissue sectionand a corresponding schematic representation (reproduced from Alberts etal., Molecular Biology of the Cell).

FIG. 6 shows a graph of FACS quantification of bone marrow (TBM) andstroma cells (MSC) for level of expression of ChroM1 and visualizationby immunohistochemical staining for MSC.

FIG. 7A shows FACS quantification of MSC (forward and side scatter),estimating the cell size and cytoplasmic granulation. The resultsexpressed the ChroM1^(VE+)/ChroM1^(VE−) from the MSC divided into twosubpopulations of small (S) and large (L) cells, ChroM1^(VE+) cells arein S region (FIG. 7A). In FIG. 7B, FACS analysis of the cell cycle basedon DNA staining with PI, dot plot demonstrates double staining with PIversus membranous staining with FITC labeled anti ChroM antibody. 76%ChroM1^(VE+) cells are in GI phases and are of the small cell size, andthe ChroM1^(VE+) expression on the cell surface is assessed for cellsize and cycle.

FIG. 8A shows a graph of a FACS analysis of double staining with cellsurface ChroM1 and with intracellular cell cycle markers (c-Fos, cjun,Ki-67, cyclinD1 and B1). Data is presented as percent of positivelystained cells for ChroM1 and each of the markers (mean±SD) obtained fromat least three experiments. FIG. 8B is a schematic illustration of thepresence of the intracellular cell cycle markers at different stages inthe cell cycle.

FIGS. 9A and 9B show the results of a FACS analysis for ChroM1expression in cultured cells from four donors. 1×10⁵ MSC were plated in100 mm culture dish and grown for a week in the growth medium (10% FCSin DMEM). The plates were then replaced with fresh medium contained 2%FCS (low serum) or 10% FCS (high serum) for an additional week. Cellswere then released with EDTA and subjected to analysis by FACS forChroM1 expression.

FIG. 10 shows a graph of an analysis of cell surface markers. Theresults represent the co-expression of ChroM with one of the selectedmarkers: CD44, CD51, CD61, CD62E, CD62L, CD62P and CD34. The doublestaining analysis results are shown by histograms representing theexpression of ChroM1^(VE+) to a specific marker or negative cells. Theanalysis was performed for different donors n=6.

FIG. 11 shows a graph of ATPase activity in IP, for 3 different donors,calculated as an average±SD of triplicates in each experiment. Acalibration curve of serial concentrations of KHPO₄ was used todetermine the amount of Pi released in the colorimetric assay.

FIG. 12A shows multiple alignment of KR region (DNA binding domain ofHMGI/Y; SEQ ID NO:19) in ChroM1 (SEQ ID NO:15), BRM (SEQ ID NO:16), BRG1(SEQ ID NO:17) and CHD1 (SEQ ID NO:18) and FIG. 12B shows an EMSA gelthat represents the mobility of ³²P-labeled oligonucleotide in thepresence and absence of rP (2-1 μg, 3-3 μg 4-3μg and 100× excess ofunlabeled oligonucleotide).

FIG. 13 shows the IP results from biotinylated cell membrane and ChIPextraction on gel stained with Coomassie and onWestern blot.

FIG. 14 The ChIP analysis of primary MSC cells from 3 human donorsfollowed with PCR amplification for three promoters at two regions (P,proximal and D, distal). Lane 1 is control and lanes 2 and 3 are treatedcells in the presence of 17-β estradiol or TGFβ, respectively.

FIGS. 15A and 15B show PCR amplification from ChIP DNA for EMSA analysisof estrogen receptor promoter (ER; FIG. 15A) and BMP4 promoter (FIG.15B) where: − indicates probe only; + indicates with recombinant protein(rP); and D indicates in the presence of distamycin A.

FIGS. 16A-16G show immunohistochemical staining of mouse embryo sectionsat day 16 and day 4 post-natal. The staining is in mesenchymalcondensation during skeletal development.

FIG. 17 shows the immunohistochemistry of mouse sections where positivestaining is detected in progenitors at the skeletal muscle, which aresuggested as satellite cells.

FIGS. 18A-18D show the histopathology of normal human bone section andtumors in comparison to cultured cells. The ChroM1 expression in tissuesection of normal bone (FIG. 18A) or cultured MSC was membranous orcytoplasm (FIG. 18B). Biopsies of osteogenic sarcoma (FIG. 18C)expressed a different cellular pattern, where the protein wastranslocated to the nucleus (FIG. 18D).

FIG. 19 shows schematic illustrations of alternative splice forms forChroM1, ChroM2, and ChroM3 and hypothetical protein FLJ12178 by showingexon size and number.

FIGS. 20A-20D show schematic illustrations of open reading frames (ORFs)in different frames for the splice forms, ChroM1 (FIG. 20A), ChroM3(FIG. 20B), ChroM2 (FIG. 20C), and hypothetical protein FLJ12178 (FIG.20D). The representations are the same as presented in FIGS. 1 and19A-19D.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery by the present inventorsof a novel chromatin remodeling protein, ChroM1, which is present instromal precursor cells/osteogenic and muscle cells. Bioinformaticanalysis showed that other known proteins that have similar domainmotifs or combinations of domains are involved with steroid receptorsand in chromatin remodeling. ChroM1 has ATPase activity and DNA BindingDomain (DBD) that was found to co-precipitate with DNA. Usinganti-ChroM1 antibodies, the laboratory of the present inventors foundChroM1 by FACS and by immunohistochemistry in osteoblastic cells at thecell membrane and in the cytoplasm with negligible amounts in thenucleus. It is expected that ChroM1 is a co-factor that is part of acomplex system of chromatin regulation in association with the steroidhormones (SR) such as estrogen and glucocorticoid receptors that allowthe opening up of chromatin for access by the steroid and for initiatingthe start of the transcription process. Transcription factors haverestricted access to DNA, and they are affected locally by chromatinremodeling factors, which have a key role in differential geneexpression.

In addition, it appears that the ChroM1 protein is part of the SWI/SNF2subfamily, which is known to be a marker of embryonic development. Fromthe results obtained in mouse development, it was found that ChroM1 as amarker appears to be directly related to skeletal development. It isknown that skeletal development begins at 16.5 days in the mouse, andthe presence of the marker coincides exactly with the beginning ofskeletal development.

The laboratory of the present inventors has also found that this ChroM1marker is present in proliferating cells but is either absent or notpredominantly present in resting cells. ChroM1 is expressed with othermarkers of early differentiation such as CD34, CD44, selectin receptorsand integrin receptors.

ChroM1 has the amino acid sequence of SEQ ID NO:2. While ChroM1 is apreferred embodiment of the polypeptide according to the presentinvention, it is intended that the polypeptide comprehend a fragment ofChroM1, a variant of ChroM1 having at least 95% sequence identity,preferably at least 98% sequence identity, to the amino acid sequence ofSEQ ID NO:2, and a fragment of this variant provided that such apolypeptide has the activity of a chromatin remodeling protein, i.e.,ATPase activity and DNA binding domain, etc.

Naturally-occurring alternative splice variants of ChroM1 were alsodiscovered by the inventor and are considered to be part of the presentinvention. Accordingly, the present invention includes three specificnaturally occurring alterative splice variants ChroM2 (SEQ ID NO:4),ChroM3 (SEQ ID NO:6), and a hypothetical protein FLJ12178 (SEQ ID NO:8).

One aspect of the present invention also relates to a composition whichcontains one or a combination of the polypeptide of the presentinvention and the naturally-occurring alternative splice variantsthereof. This composition further includes an excipient, diluent,carrier or auxiliary agent, which is preferably pharmaceuticallyacceptable.

The present invention is also directed to a molecule having theantigen-binding portion of an antibody which binds to a polypeptideaccording to the present invention. The polypeptide of the presentinvention is preferably ChroM1 and the binding is preferably with highspecificity.

It should be understood that when the terms “antibody” or “antibodies”are used, this is intended to include intact antibodies, such aspolyclonal antibodies or monoclonal antibodies (mAbs), as well asproteolytic fragments thereof such as the Fab or F(ab′)₂ fragments.Furthermore, the DNA encoding the variable region of the antibody can beinserted into the DNA encoding other antibodies to produce chimericantibodies (see, for example, U.S. Pat. No. 4,816,567). Single chainantibodies can also be produced and used. Single chain antibodies can besingle chain composite polypeptides having antigen binding capabilitiesand comprising a pair of amino acid sequences homologous or analogous tothe variable regions of an immunoglobulin light and heavy chain (linkedV_(H)-V_(L) or single chain F_(V)). Both V_(H) and V_(L) may copynatural monoclonal antibody sequences or one or both of the chains maycomprise a CDR-FR construct of the type described in U.S. Pat. No.5,091,513, the entire contents of which are hereby incorporated hereinby reference. The separate polypeptides analogous to the variableregions of the light and heavy chains are held together by a polypeptidelinker. Methods of production of such single chain antibodies,particularly where the DNA encoding the polypeptide structures of theV_(H) and V_(L) chains are known, may be accomplished in accordance withthe methods described, for example, in U.S. Pat. Nos. 4,946,778,5,091,513 and 5,096,815, the entire contents of each of which are herebyincorporated herein by reference.

A “molecule having the antigen-binding portion of an antibody” as usedherein is intended to include not only intact immunoglobulin moleculesof any isotype and generated by any animal cell line or microorganism,but also the antigen-binding reactive fraction thereof, including, butnot limited to, the Fab fragment, the Fab′ fragment, the F(ab′)₂fragment, the variable portion of the heavy and/or light chains thereof,Fab miniantibodies (see WO 93/15210, U.S. patent application Ser. No.08/256,790, WO 96/13583, U.S. patent application Ser. No. 08/817,788, WO96/37621, U.S. patent application Ser. No. 08/999,554, the entirecontents of which are incorporated herein by reference) and chimeric orsingle-chain antibodies incorporating such reactive fraction, as well asany other type of molecule in which such antibody reactive fraction hasbeen physically inserted. Such molecules may be provided by any knowntechnique, including, but not limited to, enzymatic cleavage, peptidesynthesis or recombinant techniques.

The term “epitope” is meant to refer to that portion of any moleculecapable of being bound by an antibody which can also be recognized bythat antibody. Epitopes or antigenic determinants usually consist ofchemically active surface groupings of molecules such as amino acids orsugar side chains and have specific three-dimensional structuralcharacteristics as well as specific charge characteristics.

An “antigen” is a molecule or a portion of a molecule capable of beingbound by an antibody which is additionally capable of inducing an animalto produce antibody capable of binding to an epitope of that antigen. Anantigen may have one or more than one epitope. The specific reactionreferred to above is meant to indicate that the antigen will react, in ahighly selective manner, with its corresponding antibody and not withthe multitude of other antibodies which may be evoked by other antigens.

Monoclonal antibodies (mAbs) are a substantially homogeneous populationof antibodies to specific antigens. MAbs may be obtained by methodsknown to those skilled in the art. See, for example Kohler et al (1975);U.S. Pat. No. 4,376,110; Ausubel et al (1987-1999); Harlow et al (1988);and Colligan et al (1993), the contents of which references areincorporated entirely herein by reference. Such antibodies may be of anyimmunoglobulin class including IgG, IgM, IgE, IgA, and any subclassthereof. The hybridbma producing the mAbs of this invention may becultivated in vitro or in vivo. High titers of mAbs can be obtained inin vivo production where cells from the individual hybridomas areinjected intraperitoneally into pristane-primed Balb/c mice to produceascites fluid containing high concentrations of the desired mAbs. MAbsof isotype IgM or IgG may be purified from such ascites fluids, or fromculture supernatants, using column chromatography methods well known tothose of skill in the art.

Chimeric antibodies are molecules, the different portions of which arederived from different animal species, such as those having a variableregion derived from a murine mAb and a human immunoglobulin constantregion. Antibodies which have variable region framework residuessubstantially from human antibody (termed an acceptor antibody) andcomplementarity determining regions substantially from a mouse antibody(termed a donor antibody) are also referred to as humanized antibodies.Chimeric antibodies are primarily used to reduce immunogenicity inapplication and to increase yields in production, for example, wheremurine mAbs have higher yields from hybridomas but higher immunogenicityin humans, such that human/murine chimeric mAbs are used. Chimericantibodies and methods for their production are known in the art (Betteret al, 1988; Cabilly et al, 1984; Harlow et al, 1988; Liu et al, 1987;Morrison et al, 1984; Boulianne et al, 1984; Neuberger et al, 1985;Sahagan et al (1986); Sun et al, 1987; Cabilly et al, European PatentApplication 125023 (published Nov. 14, 1984); Taniguchi et al, EuropeanPatent Application 171496 (published Feb. 19, 1985); Morrison et al,European Patent Application 173494 (published Mar. 5, 1986); Neubergeret al, PCT Application WO 8601533, (published Mar. 13, 1986); Kudo etal, European Patent Application 184187 (published Jun. 11, 1986);Morrison et al., European Patent Application 173494 (published Mar. 5,1986); and Robinson et al., International Patent Publication WO 9702671(published May 7, 1987) Queen et al., (1989) and WO 90/07861, U.S. Pat.Nos. 5,693,762, 5,693,761, 5,585,089, 5,530,101 and Winter, U.S. Pat.No. 5,225,539, and WO 92/22653. These references are hereby incorporatedby reference.

Besides the conventional method of raising antibodies in vivo,antibodies can be produced in vitro using phage display technology. Sucha production of recombinant antibodies is much faster compared toconventional antibody production and they can be generated against anenormous number of antigens. By contrast, in the conventional method,many antigens prove to be non-immunogenic or extremely toxic, andtherefore cannot be used to generate antibodies in animals. Moreover,affinity maturation (i.e., increasing the affinity and specificity) ofrecombinant antibodies is very simple and relatively fast. Finally,large numbers of different antibodies against a specific antigen can begenerated in one selection procedure. To generate recombinant monoclonalantibodies one can use various methods all based on phage displaylibraries to generate a large pool of antibodies with different antigenrecognition sites. Such a library can be made in several ways: One cangenerate a synthetic repertoire by cloning synthetic CDR3 regions in apool of heavy chain germline genes and thus generating a large antibodyrepertoire, from which recombinant antibody fragments with variousspecificities can be selected. One can use the lymphocyte pool of humansas starting material for the construction of an antibody library. It ispossible to construct naive repertoires of human IgM antibodies and thuscreate a human library of large diversity. This method has been widelyused successfully to select a large number of antibodies againstdifferent antigens. Protocols for bacteriophage library construction andselection of recombinant antibodies are provided in the well-knownreference text Current Protocols in Immunology, Colligan et al (Eds.),John Wiley & Sons, Inc. (1992-2000), Chapter 17, Section 17.1.

Due to the discovery that ChroM1 is present in human osteogenicprogenitor cells and proliferating osteoblasts, but not in matureosteoblasts, ChroM1 can be used as a marker for distinguishing thepartially differentiated osteogenic progenitor cells and proliferatingosteoblasts from mature osteoblasts and other non-ChroM-containingcells. For instance, bone marrow, which is the soft tissue occupying themedullary cavities of long bones, some haversian canals, and spacesbetween trabeculae of cancellous or spongy bone, is a source ofosteogenic progenitor cells and proliferating osteoblasts. However, inorder to isolate an enriched population of osteogenic progenitor cellsand proliferating osteoblasts from a cell mixture obtained from bonemarrow, a procedure for selectively recovering the osteogenic progenitorcells and proliferating osteoblasts is needed.

Osteogenic progenitor cells and proliferating osteoblasts can beseparated from other cells by virtue of the presence of ChroM1 expressedon these cells. A molecule containing the antigen binding portion of anantibody specific for ChroM1, which molecule is preferably a polyclonalanti-ChroM1 antibody, can be used to enrich for a cell population ofChroM1 positive osteogenic progenitor cells and proliferatingosteoblasts from a cell mixture. Thus, the present invention provides amethod for separating a cell population containing human osteogenicprogenitor cells and proliferating osteoblasts from a cell mixturederived from a source of these cells, such as bone marrow. This methodinvolves contacting the cell mixture with a molecule having the antigenbinding portion of an antibody specific for ChroM1, which moleculeselectively binds to a ChroM1 antigen on human osteogenic progenitorcells and proliferating osteoblasts. The antibody-bound cells are thenseparated from the cell mixture.

The cells can be isolated by conventional techniques for separatingcells, such as those described in Civin, U.S. Pat. Nos. 4,714,680,4,965,204, 5,035,994, and 5,130,144, Tsukamoto et al 5,750,397, andLoken et al, U.S. Pat. No. 5,137,809, all of which are herebyincorporated by reference in their entirety. Thus, for example, aChroM1-specific polyclonal antibody can be immobilized, such as on acolumn or on magnetic beads. The entire cell mixture may then be passedthrough the column or added to the magnetic beads. Those which remainattached to the column or are attached to the magnetic beads, which maythen be separated magnetically, are those cells which contain a markerwhich is recognized by the antibody used. Thus, if the anti-ChroM1antibody is used, then the resulting population will be greatly enrichedin ChroM1 positive cells.

Another way to sort progenitor cells is by means of flow cytometry, mostpreferably by means of a fluorescence-activated cell sorter (FACS), suchas those manufactured by Becton-Dickinson under the names FACScan orFACSCalibur. By means of this technique, the cells having a ChroM1marker thereon are tagged with a particular fluorescent dye by means ofan anti-ChroM1 antibody (or more generally a molecule having the antigenbinding of an anti-ChroM1 antibody) which has been conjugated to such adye. Similarly, another marker for a specific subpopulation ofosteogenic progenitor cells are tagged with a different fluorescent dyeby means of an antibody against this second marker which is conjugatedto the other dye. When the stained cells are placed on the instrument, astream of cells is directed through an argon laser beam that excites thefluorochrome to emit light. This emitted light is detected by aphoto-multiplier tube (PMT) specific for the emission wavelength of thefluorochome by virtue of a set of optical filters. The signal detectedby the PMT is amplified in its own channel and displayed by a computerin a variety of different forms e.g., a histogram, dot display, orcontour display. Thus, fluorescent cells which emit at one wavelength,express a molecule that is reactive with the specificfluorochrome-labeled reagent, whereas non-fluorescent cells orfluorescent cells which emit at a different wavelength do not expressthis molecule but may express the molecule which is reactive with thefluorochrome-labeled reagent which fluoresces at the other wavelength.The flow cytometer is also semi-quantitative in that it displays theamount of fluorescence (fluorescence intensity) expressed by the cell.This correlates, in a relative sense, to the number of the moleculesexpressed by the cell.

Flow cytometers are also equipped to measure non-fluorescent parameters,such as cell volume or light scattered by the cell as it passes throughthe laser beam. Cell volume is usually a direct measurement. The lightscatter PMTs detect light scattered by the cell either in a forwardangle (forward scatter; FSC) or at a right angle (side scatter; SSC).FSC is usually an index of size, whereas SSC is an index of cellularcomplexity, although both parameters can be influenced by other factors.

Preferably, the flow cytometer is equipped with more than one PMTemission detector. The additional PMTs may detect other emissionwavelengths, allowing simultaneous detection of more than onefluorochrome, each in individual separate channels. Computers allow theanalysis of each channel or the correlation of each parameter withanother. Fluorochromes which are typically used with FACS machinesinclude fluorescein isothiocyanate (FITC), which has an emission peak at525 nm (green), R-phycoerythrin (PE), which has an emission peak at 575nm (orange-red), propidium iodide (PI), which has an emission peak at620 nm (red), 7-aminoactinomycin D (7-AAD), which has an emission peakat 660 nm (red), R-phycoerythrin Cy5 (RPE-Cy5), which has an emissionpeak at 670 nm (red), and allophycocyanin (APC), which has an emissionpeak at 655-750 nm (deep red).

These and other types of FACS machines may have the additionalcapability to physically separate the various fractions by deflectingthe cells of different properties into different containers.

Any other method for isolating the osteogenic progenitor cells as anenriched population from a cell mixture as a starting material, such asbone marrow, may also be used in accordance with the present invention.The various subpopulations of the present invention may be isolated in asimilar manner.

Certain additional markers can be used to differentiate betweensubpopulations of osteogenic progenitor cells and proliferatingosteoblasts, such as CD44, CD51, CD61, CD62 (E, L, P), CD34, etc.

By contacting either enriched anti-ChroM antibody-bound cells or thestarting cell mixture with a second antibody which selectively binds asecond antigen in a subpopulation of human osteogenic progenitor cellsand proliferating osteoblasts, the subpopulation of human osteogenicprogenitor cells can be separated following a first enrichment forChroM1 positive cells.

A further aspect of the present invention is directed to an isolatedcell population enriched for human stromal progenitors. For example,proliferating osteoprogenitors, preferably obtained using the methods ofseparating progenitor cells and proliferating osteoblasts from a cellmixture according to the present invention. The isolated cell populationmay be additionally enriched for those progenitor cells that candifferentiate into muscle cells. These enriched cell populations may beused for tissue engineering of skeletal muscle, cardiac and bonetissues. Preferably, the human osteogenic progenitor cells that candifferentiate into osteogenic cells and muscle cells according to thepresent invention are not human embryonic stem cells.

While osteogenic progenitor cells and proliferating osteoblasts can beisolated in substantial purity, i.e., in a substantially homogeneouspopulation greater than 50%, preferably greater than 60%, morepreferably greater than 70% cells which are ChroM1 positive, by themethods discussed above, such as, for example, by means of the FACSapparatus, it is not always necessary that the ChroM1 positiveosteogenic and proliferating osteoblast cell population of the presentinvention be present in substantial purity. For example, the presentinvention also comprehends an isolated population of cells containinggreater than 40% cells which are positive for ChroM1. Normally, cellpopulations containing human osteogenic progenitor cells sampled fromthe body contain less than 10% cells positive for ChroM1. Such a lowpurity subpopulation still defines over the prior art and yet maintainsmany of the advantages of the present invention. Isolated cellpopulations having greater than 30% of ChroM1 positive cells are alsoconsidered to be part of the present invention. A further aspect of thepresent invention is directed to a composition which contains aphysiologically acceptable medium and the isolated cell population ofcells according to the present invention.

The isolated or separated human osteogenic progenitor cells andproliferating osteoblasts of the present invention, whether or notobtained using the method according to the present invention, can beexpanded in number by long term in vitro culture with minimaldifferentiation.

The pluripotent progenitors can be induced to differentiate undervarious culture conditions.

The localization of ChroM1 in different cellular compartments such asthe cell membrane, cytoplasm, and the nucleus provides a connectionbetween the extracellular environment and transcription. Tissue specificprogenitor cells can proliferate in vivo in the appropriate niche only.Thus in vitro expanded mesenchymal cells can be used for the recovery ofdefects in bone, skeletal and cardio-muscle. This may be affected by theimmediate regulation and genomic effect of steroid hormones in a tissueand cell specific manner.

After population expansion in culture, isolated stromal progenitor cellscan then be harvested and activated to differentiate under variousconditions, such as mechanical, cellular, and biochemical stimuli. Byactivating stromal progenitors to differentiate into the specific typesof cells desired, such as bone-forming osteoblast cells, etc., a highlyeffective process exists for treating skeletal and other connectivetissue disorders.

The culture medium can also contain additional components, such asosteoinductive factors. The osteoinductive factors include any that arenow known and any factors which are later recognized to haveosteoinductive activity. Such osteoinductive factors include, forexample, dexamethasone, ascorbic acid-2-phosphate, β-glycerophosphateand TGF superfamily proteins, such as the bone morphogenic proteinsBMPS. The presence of bioactive factors such as dexamethasone, ascorbicacid-2-phosphate and β-glycerophosphate in the culture medium directshuman MSCs into the osteogenic lineage. The presence of a bioactivefactor such as 5-azacytidine, 5-azadeoxycytidine, or analogs of eitherof them in the culture medium directs human mesenchymal stem cells intothe myogenic lineage.

As a result, a process has been developed for isolating and purifyinghuman osteogenic progenitor cells from tissue prior to differentiationand then culture expanding the osteogenic progenitor cells to produce avaluable tool for musculoskeletal therapy. The objective of suchmanipulation is to greatly increase the number of osteogenic progenitorcells and to utilize these cells to redirect and/or reinforce the body'snormal reparative capacity. The osteogenic progenitor cells are expandedto great numbers and applied to areas of connective tissue damage toenhance or stimulate in vivo growth for regeneration and/or repair, toimprove implant adhesion to various prosthetic devices throughsubsequent activation and differentiation, etc.

Along these lines, various procedures are contemplated, as would be wellappreciated by those of skill in the art, for transferring,immobilizing, and activating the culture-expanded, purified osteogenicprogenitor cells at the site for repair, implantation, etc., includinginjecting the cells at the site of a skeletal defect, incubating thecells with a prosthesis and implanting the prosthesis, etc. Thus, byisolating, purifying and greatly expanding the number of cells prior todifferentiation and then actively controlling the differentiationprocess by virtue of their positioning at the site of tissue damage orby pre-treating in vitro prior to their transplantation, theculture-expanded, osteogenic progenitor cells can be utilized forvarious therapeutic purposes such as to alleviate cellular, molecular,and genetic disorders in a wide number of metabolic bone diseases,skeletal dysplasias and other musculoskeletal and connective tissuedisorders.

In the context of tissue engineering and skeletal and cardiac tissuerepair, tissue regeneration therapy is the local application ofautologous (host-derived) and allogeneic (non-host derived) cells topromote reconstruction of tissue defects caused by trauma, disease orsurgical procedures. The objective of the tissue regeneration therapyapproach is to deliver high densities of repair-competent cells (orcells that can become competent when influenced by the localenvironment) to the defect site in a format that optimizes both initialwound mechanics and eventual neo-tissue production. For soft tissuerepair, it is likely that an implant vehicle(s), such as a matrix orscaffold, will be required to 1) transport and constrain the autologouscells in the defect site and 2) provide initial mechanical stability tothe surgical site. In an optimal system, it is likely that the vehiclewill slowly biodegrade at a rate comparable to the production ofneo-tissue and development of strength in the reparative tissue(Goodship et al., 1986).

One aspect of the present invention provides a method of treating boneor muscle tissue damage involving administering to a patient in needthereof with bone or muscle tissue damage the isolated population ofhuman cells enriched for human osteogenic progenitor cells according tothe present invention. These cells are preferably culture expanded andare preferably autologous to the patient to which they are administered.In addition, the administered cells can be administered as part of animplant vehicle such as a matrix or scaffold for tissue regeneration.

A further aspect of the present invention provides a method ofgenerating bone or muscle tissue, such as cardio-muscle, which involvesseeding a matrix or scaffold, that can be used as an implant vehicle ina patient, with the isolated population of cells enriched for humanosteogenic progenitor cells according to the present invention. Theseeded cells are preferably culture expanded either before or afterseeding the matrix or scaffolding. Non-limiting examples of matrices orscaffolding suitable for tissue engineering/regeneration are taught inU.S. Pat. Nos. 6,365,149; 6,200,606; 5,939,323; 6,323,146; 6,323,278;5,893,888; and 6,228,117, the contents of which are herein incorporatedby reference.

A still further aspect of the invention relates to the discovery thatChroM1 is translocated to the nucleus in osteogenic sarcoma cells.Therefore osteosarcoma cells can be identified based on staining ofChroM1 in the nuclei of osteosarcoma cells using anti-ChroM1 antibodiesto indicate their degree of malignancy; the nuclei in osteosarcoma cellsare darkly stained using anti-ChroM1 antibodies. By contrast, the nucleiof non-osteosarcoma cells are not stained. As will be appreciated bythose in the art, this discovery can also be extended to usingdifferential staining with anti-ChroM1 antibodies to the nuclei ofosteosarcoma cells in a method for evaluating the effectiveness of atreatment for osteogenic sarcoma. The method involves contacting atissue sample, i.e., biopsy, containing osteogenic cells with a moleculehaving an antigen-binding portion of an anti-ChroM1 antibody anddetecting the presence of the molecule in the nuclei of cells in thetissue sample to identify osteosarcoma cells and to evaluate theeffectiveness of the treatment.

A further aspect of the present invention is directed to a nucleic acidmolecule containing a nucleotide sequence encoding any of thepolypeptides according to the present invention, which preferablycomprise the amino acid sequence of SEQ ID NO:2 (ChroM1), or thespecific splice variant polypeptides ChroM2 (SEQ ID NO:4), ChroM3 (SEQID NO:6), and hypothetical protein FLJ 12178 (SEQ ID NO:8), ahypothetical alternative ChroM splice form. More specifically, thenucleic acid molecule contains a nucleotide sequence coding for ChroMwhich corresponds to nucleotides 88 to 8781 of SEQ ID NO:1, or anucleotide sequence coding for any of the alternative splice variantscorresponding to nucleotide 447 to 3728 of SEQ ID NO:3, nucleotide 402to 1298 of SEQ ID NO:5, or nucleotide 196 to 1206 of SEQ ID NO:7.

Also comprehended by the present invention are nucleic acid moleculesencoding alternative splice variants which are based on computeranalysis and cloning by RT-PCR (Table 10), and nucleic acid moleculeswhich hybridizes to the nucleotide sequence corresponding to 88 to 8781of SEQ ID NO:1 under high stringency conditions.

Stringency conditions are a function of the temperature used in thehybridization experiment and washes, the molarity of the monovalentcations in the hybridization solution and in the wash solution(s) andthe percentage of formamide in the hybridization solution. In general,sensitivity by hybridization with a probe is affected by the amount andspecific activity of the probe, the amount of the target nucleic acid,the detectability of the label, the rate of hybridization, and theduration of the hybridization. The hybridization rate is maximized at aTi (incubation temperature) of 20-25° C. below Tm for DNA:DNA hybridsand 10-15° C. below Tm for DNA:RNA hybrids. It is also maximized by anionic strength of about 1.5M Na⁺. The rate is directly proportional toduplex length and inversely proportional to the degree of mismatching.

Specificity in hybridization, however, is a function of the differencein stability between the desired hybrid and “background” hybrids. Hybridstability is a function of duplex length, base composition, ionicstrength, mismatching, and destabilizing agents (if any).

The Tm of a perfect hybrid may be estimated for DNA:DNA hybrids usingthe equation of Meinkoth et al (1984), asTm=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/Land for DNA:RNA hybrids, asTm=79.8° C.+18.5 (log M)+0.58 (% GC)−11.8 (% GC)²−0.56(% form)−820/Lwhere

-   -   M, molarity of monovalent cations, 0.01-0.4 M NaCl, % GC,        percentage of G and C nucleotides in DNA, 30%-75%, % form,        percentage formamide in hybridization solution,    -   and L, length hybrid in base pairs.

Tm is reduced by 0.5-1.5° C. (an average of 1° C. can be used for easeof calculation) for each 1% mismatching.

The Tm may also be determined experimentally. As increasing length ofthe hybrid (L) in the above equations increases the Tm and enhancesstability, the full-length-rat gene sequence can be used as the probe.

Filter hybridization is typically carried out at 68° C., and at highionic strength (e.g., 5-6×SSC), which is non-stringent, and followed byone or more washes of increasing stringency, the last one being of theultimately desired high stringency. The equations for Tm can be used toestimate the appropriate Ti for the final wash, or the Tm of the perfectduplex can be determined experimentally and Ti then adjustedaccordingly.

Hybridization conditions should be chosen so as to permit allelicvariations, but avoid hybridizing to other genes. In general, stringentconditions are considered to be a Ti of 5° C. below the Tm of a perfectduplex, and a 1% divergence corresponds to a 0.5-1.5° C. reduction inTm. Typically, rat clones were 95-100% identical to database ratsequences, and the observed sequence divergence may be artifactual(sequencing error) or real (allelic variation). Hence, use of a Ti of5-15° C. below, more preferably 5-10° C. below, the Tm of the doublestranded form of the probe is recommended for probing a rat cDNA librarywith a rat DNA probe or a human cDNA library with a human DNA probe.

As used herein, highly stringent conditions are those which are tolerantof up to about 5% sequence divergence. Without limitation, examples ofhighly stringent (5-10° C. below the calculated Tm of the hybrid) andmoderately stringent (15-10° C. below the calculated Tm of the hybrid)conditions use a wash solution of 0.1×SSC (standard saline citrate) and0.5% SDS at the appropriate Ti below the calculated Tm of the hybrid.The ultimate stringency of the conditions is primarily due to thewashing conditions, particularly if the hybridization conditions usedare those which allow less stable hybrids to form along with stablehybrids. The wash conditions at higher stringency then remove the lessstable hybrids. A common hybridization condition that can be used withthe highly stringent to moderately stringent wash conditions describedabove is hybridization in a solution of 6×SSC (or 6×SSPE), 5× Denhardt'sreagent, 0.5% SDS, 100 μg/ml denatured, fragmented salmon sperm DNA atan appropriate incubation temperature Ti.

The present invention also provides a vector containing the nucleic acidmolecule according to the present invention and a host cell transformedwith the nucleic acid molecule of the present invention. Moreover, thepresent invention provides a process for producing or preparing apolypeptide or a variant thereof according to the present invention.This process involves artificially/recombinantly expressing thepolypeptide or a variant thereof in a recombinant host cell, where thenucleotide sequence encoding the polypeptide or a variant thereof isoperably-linked to a promoter suitable for driving expression of thepolypeptide or variant thereof in the host cell. The expressedpolypeptide is then recovered to prepare the polypeptide or variantthereof.

The present invention further provides the identification of the genomiclocalization for ChroM. The ChroM gene was mapped to chromosome 16q12.2using the radiation hybrid panel RH-G3 (Stanford Human Genome Center).The localization was confirmed with the advancement of the human genomeproject and localized to the contigs AC007345, AC007906. It would bewell appreciated by those of skill in the art that the genomic ChroMgene can be isolated based on this genome localization.

Overexpression/overproduction of ChroM1 may contribute to an abnormalityfound in the disease osteopetrosis. Accordingly, an aspect of thepresent invention also relates to a method for treating osteopetrosis byinhibiting the production (overproduction) of ChroM1. A preferredembodiment of this method involves administering an antisenseoligonucleotide, which inhibits production of ChroM1 and its activity asa chromatin remodeling protein, to a patient in need thereof. Theantisense oligonucleotide is complementary to a messenger RNA (mRNA),encoding ChroM1 which includes nucleotides 88 to 8781 of SEQ ID NO:1.

The ChroM1 protein of the present invention is involved with genomeintegrity and the control of chromatin remodeling complexes thatfacilitate gene expression by helping transcription factors gain accessto their DNA targets in chromatin. The present inventors believe thatChroM1 plays a role in the assembly and activity of the nuclear receptor(NR) transcription complex and serves as a co-factor which is part of acomplex system of chromatin regulation in association with steroidhormone receptors (SR), such as estrogen and glucocorticoid receptors,that allows opening up of chromatin for access by the steroid and forinitiating the start of the transcription process.

It is also believed that reduced expression of ChroM1 may contribute toa reduction in osteogenic progenitor cell activity, such as found inosteoporosis. Accordingly, another aspect of the present inventionrelates to a method for treating osteoporosis by inducing the expressionof ChroM1. A preferred embodiment of this method involvespharmacological modulation to induce or enhance production of ChroM1 andits activity as a chromatin remodeling protein, such as by administeringto a patient in need thereof a compound that enhances/induces theexpression of ChroM1 as screened and identified by the method describedbelow.

The present invention provides for a method of screening for andidentifying an enhancer or inhibitor compound that affects expression ofthe ChroM1 chromatin remodeling protein. This method involves incubatinga human cell, which expresses the ChroM1 polypeptide of SEQ ID NO:2, inthe presence or absence of a potential enhancer or inhibitor compoundthat affects expression of ChroM1. The potential enhancer or inhibitorcompound screened may be screened from among the compounds in chemicallibraries such as are available at many pharmaceutical companies. Afterincubation, the level of expression of ChroM1 in the presence of thepotential enhancer or inhibitor compound is determined relative to thelevel of expression of ChroM1 in the absence of the potential enhanceror inhibitor compound. A screened potential enhancer or inhibitorcompound is identified as an enhancer compound if the level ofexpression of ChroM1 in the presence of the potential enhancer compoundis substantially more than that in the absence of the potential enhancercompound. Conversely, a screened potential enhancer or inhibitorcompound is identified as an inhibitor compound if the level ofexpression of ChroM1 in the presence of the potential inhibitor compoundis substantially less than that in the absence of the potentialinhibitor compound. The identified enhancer or inhibitor compound canfurther be isolated once it is identified.

While a compound that ehances the expression of ChroM1 can be used totreat osteoporosis, a compound that inhibits the expression of ChroM1,such as identified by the method described above can be used, forinstance, to treat osteopetrosis or osteoscarcoma by administering theinhibitor compound to a patient in need thereof.

As shown in the Example, ChroM1 was demonstrated to bind promoters ofgenes that play an important role in bone biology. This provides fordirectly manipulating the activity of these genes by using a compoundthat affects the binding of ChroM1 to the promoters of these genes,which may result in a restricted and directed effect on key boneproteins in a tissue and time-specific manner. A number of genes thatplay an important role in bone biology, i.e., osteoblast cell functionand differentiation, and their promoters have been identified in theprior art. The laboratory of the present inventors have used thepromoters of three genes, estrogen receptor α (ERα; Genbank accessionno. X63118), bone morphogenic protein-4 (BMP-4; Genbank accession no.U43842; van de Wijngaard et al., 2000), and osteocalcin (Lian et al.,1998) for binding studies with ChroM1. The studies of ChroM1 binding topromoters as disclosed in the Example show that in a chromatinimmunoprecipitation (ChIP) assay, the DNA binding domain (DBD) of ChroM1binds to the A/T-rich distal promoter region of ERα and BMP-4. ChroM1binding was reduced in the presence of distamycin A, an antibiotic thatblocks binding to A/T-rich regions.

In another aspect of the present invention, a method of identifyingA/T-rich promoter regions of genes involved in modulation of osteoblastdifferentiation and which are capable of binding to the DNA bindingdomain of ChroM1 is provided. This method can be used to identify apromoter of a known gene or of an unknown gene. For instance, amicroarray of human genomic DNA fragments, as is believed to becommercially available and is believed to be within the skill of thosein the art to produce, can be contacted with a peptide containing theDNA binding domain of ChroM1 to identify A/T rich promoter regions ofpotential genes involved in modulation of osteoblast-differentiation.The method involves contacting fragments of genomic DNA with a peptidecontaining a DNA binding domain comprising residues 2429-2437,preferably comprising residues 2333-2480, of the ChroM1 protein of SEQID NO:2 and identifying a fragment of genomic DNA bound by this peptide.The nucleotide sequence of the promoter region on the fragment bound bythe peptide is determined so that the A/T-rich promoter region of a geneinvolved in modulation of osteoblast differentiation is identified.

ChroM1 is believed to be a chromatin remodeling protein that opens upchromatin to allow access to a promoter region where the DNA bindingdomain (DBD) of the ChroM1 protein can bind an AT-rich promoter regionof a gene involved in osteoblast differentiation (from osteogenicprogenitor cells to osteoblasts and muscle cells) in the presence of amodulator compound, i.e., estradiol for estrogen receptor promoter, toinitiate transcription. Thus, ChroM1 functions in transcriptionalregulation of genes involved in osteoblast differentiation in thepresence of a modulator that can be screened from chemical libraries,such as libraries of pharmacological compounds and metabolites.

The present invention further provides for a method of screening for andidentifying a compound which stimulate differentiation of osteogenicprogenitor cells by using chromatin immunoprecipitation (ChIP) withChroM1 and anti-ChroM1 antibodies followed by analysis of PCRamplification products of the DNA from the immunoprecipitated chromatin.The general ChIP method is disclosed in Orlando (2000) and Chen et al(1999). The present method using ChIP involves incubating human cells,which express the ChroM1 chromatin remodeling protein of SEQ ID NO:2with a potential stimulator compound and adding formaldehyde to theincubated human cells to crosslink proteins to DNA in chromatin by invivo fixation. Once the proteins are crosslinked to DNA in chromatin,the crosslinked chromatin is sonicated to solubilize the crosslinkedchromatin. The solubilized crossliked chromatin is thenimmunoprecipitated with antibodies specific for the ChroM1 protein ofSEQ ID NO:2 to form immunocomplexes. DNA from the immunocomplexes arerecovered and incubated under amplification conditions witholigonucleotide primers capable of amplifying an A/T-rich promoterregion of a gene involved in control of osteoblast cell differentiationif present in the recovered DNA. For example, oligonucleotide nucleotideprimers Erp3 (SEQ ID NO:28) and Erp4 (SEQ ID NO:29) were used inexperiments in the Example to amplify a 204 bp ERα A/T-rich promoterregion (SEQ ID NO:32) from the DNA recovered from ChIP. Likewise,oligonucleotide primers BMPpr3 (SEQ ID NO:20) and BMPpr4 (SEQ ID NO:21)were used in the experiments in the Example to amplify a 145 bp BMP-4A/T-rich promoter region (SEQ ID NO:33) from the DNA recovered fromChIP. If an amplication product is detected which corresponds to the A/Trich promoter region capable of being amplified with the oligonucleotideprimer used, then the potential stimulator compound is identified as astimulator compound. Besides A/T-rich promoter regions of human estrogenreceptors (i.e., ERα), human bone morphogenic protein (i.e., BMP-4), orosteocalcin, promoters of other known genes involved in osteoblast cellfunction and differentiation and promoters identified in the method ofidentifying A/T-rich promoter regions according to the present inventioncan be used with the appropriate oligonucleotide primers once thenucleotide sequences of the A/T-rich promoter regions are known.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way-of illustration and is not intended to be limiting ofthe present invention.

EXAMPLE

The laboratory of the present inventors has cloned and identified a geneof interest, designated ChroM1, that is expressed by pre-osteoblasticcells. The comparison of this gene to the genome database with a varietyof algorithms, combined with dry and wet biology, deepens the knowledgeon the structure and function of the genes. The ChroM gene was mapped onhuman chromosome 16 and the genomic structure with intron-exonboundaries that include the full-length gene structure and alternativesplicing forms was determined. The protein is involved with genomeintegrity and the control of chromatin-remodeling complexes thatfacilitate gene expression by helping transcription factors gain accessto their targets in chromatin. ChroM is related to the SWI/SNF familymembers from yeast but it is distinguished from these family membersbecause it contains a chromodomain and possesses regions that link theprotein to the cell membrane. In this example, evidence is presentedthat the identified human unique ChroM harbors structural motifs at theN-terminus of chromo domain followed by a SNF2-like domain that ischaracteristic of AAA-ATPases, DEXDc-helicases super family, and TCHregion preference for A/T rich regions. These motifs have homology tofunctional proteins that control cell growth. ChroM is expressed inproliferating cells and is correlated with the G1 phase. The proteincontains a signature motif LXXLL (NR box) that is necessary andsufficient to permit interaction with nuclear receptors.

Immunohistochemistry and FACS studies revealed that ChroM1 localizes atthe cell membrane and cytoplasm of osteoprogenitor cells and the proteinis translocated to the nucleus for its activity and is enriched in thenucleus of cancer cells such as osteosarcoma. The data presented belowsuggest that ChroM represent a novel family of membrane factors that actalso in chromatin-remodeling complexes. No other protein resembling thisfamily has yet been identified. This unique protein however has someconserved functional regions from yeast to human.

Material and Methods

In vitro culture: Human bone marrow stromal cells (MSC) were collectedfrom surgical aspirates of bone marrow (normal donors male and female atage 2.7 to 49 years) to prepare ex vivo culture plated at low-density(1.5×10⁴ cells/cm²). Clonal populations of human bone marrow stromalcells were obtained from the single cell derived cells (previouslydescribed in Shur et al. 2000). Human trabecular bone cultures (TBC)were established from fresh trabecular bone explants from femoral headsduring orthopaedic procedures. Bone samples obtained from 14 donors aged60-80 years were cultured. Primary marrow stromal cells (MSC) andtranbecularbone cells (TBC) were cultured in Dulbecco's ModifiedEssential Medium (DMEM) with the addition of 10% heat-inactivated fetalcalf serum (FCS). The growth medium for clonal populations of MSC wassupplemented with 10⁸M dexamethasone (Dex) (Ikapharm, Israel) and 10⁻⁴ML-ascorbic acid phosphate magnesium salt (Sigma, Israel).

Gene expression analysis of MSC: Total RNA was extracted from culturedcells (EZ RNA kit, Biological industries, Bet-Haemek, Israel). The RNAwas reverse transcribed using avian myeloblastosis virus reversetranscriptase (AMV-RT) and oligo-dT, in order to generate cDNA. ThiscDNA then served as a template for the polymerase chain reaction (PCR)(Takara Shuzo Co. Ltd., Japan). The integrity of the RNA, the efficiencyof RT reaction and the quality of cDNA subjected to the RT-PCR weretaken into account by controls in which a transcript ofGlucose-3-Phosphate Dehydrogenase (G3PDH) was amplified using theprimers, G3PDHf: ACCACAGTCCATGCCATCAC (SEQ ID NO:9)and G3PDHrTCCACCACCCTGTTGCTGTA (SEQ ID NO:10), obtained from Clontech, Palo Alto,Calif. Genomic DNA contamination was excluded by using primers for c-Mycand TGFb1 that amplified products of different size in the genome whencompared to cDNA. The same cDNA derived from each donor and/or clone wasused for PCR analysis with specific primers ChroMf: AGCAACACAGATGTC (SEQID NO:11), ChroMr: ATCAGGAATTCCTTGAGGTTG (SEQ ID NO:12).The reactionproducts were separated by electrophoresis in 1% agarose gels (SeaKemGTG, FMC, USA) in Tris Borate EDTA (TBE) buffer. The amplified DNAfragments were stained by ethidium bromide measured for optical density(OD) by densitometry (Bio Imaging System, BIS 202D) and analyzed using“TINA” software. PCR amplification was performed at least twice andsubjected to semi-quantitative analyses by comparison of the OD of PCRproducts for ChroM normalized to the OD of co-amplified G3PDH-PCRproduct.

Immunohistochemistry: The specimens were fixed in 4% formalin in PBS(phosphate buffered saline) immediately after excision of embryos ororgan. Where needed, decalcification was performed with 10% EDTA in PBS(pH 7.4) in 4° C. for 2 days. The specimens were washed in PBS andembedded in paraffin (50-60° C.) and cut serially in 5 widths.

For staining, deparaffinization in xylene for 15 minutes and rehydrationby an alcohol gradient followed with PBS washing. Forimmunohistochemistry, the section was blocked with 10% normal goat serumin 1% BSA/PBS for 30 minutes. Immunohistochemical stains used forlocalizing various proteins on tissue from embryogenic stages ofdevelopment-in adult mice and in section from human bone. Theimmuno-detection used anti-ChroM IgG-purified antibody from rabbitserum. The signal was amplified with second antibody, goatanti-rabbit-biotin (Dako) and extravidine-peroxidase (Sigma). Thereaction was detected with the chromagen, DAB (DAB-3,3′-Diaminobenzidinetetrahydrochloride, Sigma) that creates a hard dissolving saltsedimentation after reacting with the peroxidase. For counterstain,Hematoxilin for cell nuclei staining was used.

Flow Cytometric Analysis: Cells were EDTA-released from cultures.Fluorescence Activated Cell Sorting (FACS) analyzed the single cellsuspension. The staining for surface antigens employed biotin conjugatedantibodies to CD-44, CD-51, CD-61, CD62P, CD62L, CD62E (Pharmingen).Cell cycle markers were analyzed with antibodies: KI-67 (Dako), cFOS,cJUN (Oncogene) and Cyclin B1 and Cyclin Dl using intracellular stainingprocedure. Negative control for intracellular staining employed theisotype-matched IgG (Mouse IgG1 and Rabbit IgG) at the sameconcentration as the antibody of interest. Secondary antibodies usedwere Extravidine-PE (Sigma) or FITC-conjugated-anti-rabbit (Jacksonimmune Research Laboratories). FACS staining was performed according totechnical protocols (website www.pharmingen.com). Cell Cycle was alsoanalyzed by Propidium Iodine (PI). For each antibody, 1×10⁶ cells wasused for the staining procedure. Finally, 1×10⁴ cells were quantifiedand the analysis was performed using software from Becton Dickinson.

Protein analysis, cell biotinylation, immunoprecipitation (IP)procedure, SDS-PAGE gel and Western blot analysis: These procedures andanalyses were performed according the standard protocols websitewww.protocol-online.net/Protocol.htm. Briefly, immunoprecipitation wasperformed with ChroM1 antibody (dilution 1:700) (6 mg/ml) incubatedovernight with Protein A-Sepharose beads (Sigma). The immuno-complexeswere separated on 6.5% SDS-PAGE gel for 2 hrs, then transferred for 30min to the nitrocellulose blots and probed with primary antibody diluted1:1000 for 1 hour, followed with secondary antibody goatanti-rabbit-biotin IgG (1:2000) and Extravidin Peroxidase (1:4000) fordetection with chemiluminescent substrate (Pierce).

Chromatin immunoprecipitation (ChiP): This technique offers the abilityto detect protein that binds to protein complexes bound to DNA. Thetechnique is based on formaldehyde fixation to chromatin (Orlando, 2000and Chen et al., 1999).

ATPase activity: IP protein used to determine phosphate ions releasedduring ATPase reaction when incubated at 37° C. The protein activity wasdetermined in the presence or absence of DNA primer,GCGCAATTGCGCTCGACGATTTTTTAGCGCAATTGCGC (SEQ ID NO:13), that form a stemloop structure (Bayko et al., 1988 and Muthuswami et al., 2000).

Electrophoretic mobility shift assay (EMSA): EMSA was performed betweenrecombinant protein (rP) containing the DNA binding domain (residues2333-2480 from sequence of ChroM1) and ³²P-labeled 32-meroligonucleotide containing 24 A·T nucleotides,GATCCATATATATATATATATATATATATGCA (SEQ ID NO:14). 1-3 μg of rP and100,000 cpm of ³²P-labeled oligonucleotides were used for the bindingreaction in the presence or absence of 100 fold excess coldoligonucleotide. The reaction was carried out in 20 μl of binding buffer(26 mM Hepes, pH 7.9, 2 mM MgCl₂, 40 mM KC1, 1 mM dithiothreitol and0.25% BSA). The samples were incubated for 15 min at room temperatureprior to loading in 5% native polyacrylamide gel in 0.25 Trisborate-EDTA (TBE). The gel was run for 40 min at RT and in TBE buffer.The gels were then dried and autoradiographed.

Chromosome localization: In the analysis for chromosomal genelocalization, the Stanford Human Genome center radiation hybrid (RH)panel was used. This kit provides high-resolution maps of the humangenome using a somatic-cell approach. Hamster cells containing a humanchromosome are blasted with radiation to scramble the DNA. The damagedcell is then fused with a non-irradiated hamster cell and grown into acolony of hybrids. DNA isolated from these radiation hybrid cellsprovides the background used to order STSs and to determine the distancebetween them. The distances are calculated using statistical analyses.The Stanford G3 maps were constructed using a panel of 83 whole genomeradiation hybrids (the Stanford G3 panel) and 10,478 sequence taggedsites. The sites were derived from random genomic DNA sequences,previously mapped genetic markers and expressed sequences. These mapscover the majority of the human genome, with the markers lying at anaverage distance of 500 kb apart.

Statistical analysis—Statistical analysis of differential geneexpression by RT-PCR of cultured cells derived from various donors wasperformed by ANOVA test. Results were considered significant for P<0.05.

Softwares used for bioinformatic analysis: cDNA translation to proteinwas analyzed using BLASTN from NCBI, “Smart” program (websitewww.smart.embl-heidelberg.de) Web site Goal Remarks www.expasy.ch/tools/Translation, ORF www.expasy.ch/cgi-bin/protparam Protein parametersDot.imgen.bcm.tmc.edu:9331/seq-util/seq-util.html Changing formatPsort.nibb.ac.jp/form2.html Subcellular localizationCoot.embl-heidelberg.de/SMART Functional and structural domainswww2.ebi.ac.uk/ppsearch/ Motifs with graphicswww.cbs.dtu.dk/services/NetOGlyc/ O-glycosylationazusa.proteome.bio.tuat.ac.jp/sosui/submit.html Transmembrane domainssolubility www.ch.embnet.org/software/TMPRED_form.htmlwww.biokemi.su.se/˜server/toppred2/toppredServer.cgiwww.at.embnet.org/embnet/tools/bio/PESTfind/ PEST

Protein analysis: web sites and databases. Web site Applicationwww.expasy.ch/tools/dna.html Translation, ORFwww.expasy.ch/cgi-bin/protparam Protein parameterswww.psort.nibb.ac.jp/form2.html Subcellular localizationcoot.embl-heidelberg.de/SMART Functional and structural domainswww2.ebi.ac.uk/ppsearch/ Motifswww.pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_prosite.htmlwww.cbs.dtu.dk/databases/PhosphoBase/predict/predform.htmlPhosphorylation www.cbs.dtu.dk/services/NetOGlyc/ O-glycosylationwww.ch.embnet.org/software/TMPRED_form.html Transmembrane domainswww.biokemi.su.se/˜server/toppred2/toppredServer.cgi130.237.130.31/tmap/single.htmlwww.at.embnet.org/embnet/tools/bio/PESTfind/ PEST

PROSITE is a database of protein families and domains. It hasbiologically significant sites, patterns and profiles that help toreliably identify to which known protein family (if any) a new sequencebelongs.

ProDom database has been designed as a tool to help analyze domainarrangements of proteins and protein families.

Pfam is a collection of protein families and domains. Pfam containsmultiple protein alignments and profile-HMMs of these families. Pfam isa semi-automatic protein family database, which aims to be comprehensiveas well as accurate.

Blocks are multiply aligned ungapped segments corresponding to the mosthighly conserved regions of proteins. “Block Searcher”, “Get Blocks” and“Block Maker” are aids to detection and verification of protein sequencehomology. They compare a protein or DNA sequence to a database ofprotein blocks (current version), retrieve blocks, and create newblocks, respectively.

PRINTS is a compendium of protein fingerprints. A fingerprint is a groupof conserved motifs used to characterize a protein family; itsdiagnostic power is refined by iterative scanning of OWL. Usually themotifs do not overlap, but are separated along a sequence, though theymay be contiguous in 3D-space. Fingerprints can encode protein folds andfunctionalities more flexibly and powerfully than can single motifs: thedatabase thus provides a useful adjunct to PROSITE.

Results and Discussion

A cDNA which encodes for the full length cDNA ChrOM1 extends 11337nucleotides. The chromosomal localization was analyzed using radiationhybrid panel RH-G3 (Stanford Human Genome center). The gene was mappedto chromosome 16q12.2 based on the genomic marker GDB locus D16S3137SHGC AFMa061lyb5 with LOD 6.4, targets YACs (802h4, 813e2, 922f1,959g5). PCR analysis verified the amplified product on these YACs. Thelocalization was confirmed with the advancement of the human genomeproject and localized to the contigs AC007345, AC007906. Bioinformaticanalysis of full-length cDNA with 35 exons that represent their size andorganization (FIG. 1A). The largest open reading frame (ORF) of the cDNAresult with 8694 bp, where the first stop codon in frame appears in thelast exon of 3431 bp which is translated only in the first 877 bp andcontinues with 3′ UTR sequences and the poly (A) tail. The cDNA ORFtranslation results in a protein of 2898 amino acids from the firstmethionine with the predicted protein being approximately 326 kDa inmolecular weight. Various softwares for proteins were used to localizethe ChroM1 protein to different cellular compartments (membranous,cytoplasmic and nuclear). Examples of other proteins with duallocalization are presented in Table 1. TABLE 1 Proteins shuttle andexpression of dual localization (transmembrane and nucleus) Mechanism ofintra-cellular Protein translocation References Function BAA24799Unknown Yang L et al, 2000 Focal adhesion protein demonstrated Hic-5 tobe associated with nuclear matrix and function as steroid receptorcoactivators AAF68983 Unknown Nakamura T et al, 2000 Dual localizationin nuclear speckles HuASH1 and tight junctions, a member of trithoraxgroup of genes with chromatin remodeling activity NP_002678 UnknownOuyang P. differentiation-specific desmosomal Pinin/Mema/DRS protein,nuclear phosphoprotein XP_030094 Unknown Gottardi C J et al, 1996Tight-junction associated member of the ZO-1 family of Guanilate-cyclasehomologues, nuclear localization is associated with proliferative,immature status AAG33848 Proteolytic cleavage Artavanis-Tsakonas S,Transmembrane receptor, intracellular Notch et al, 1995 domaintranslocates to the nucleus and forms a complex with transcriptionalrepressor CIZ AB019281 Nakamoto T et al., 2000 Focal adhesion andnucleus EGF receptor Ref Transmembrane receptor translocates to thenucleus

For ChroM1 it was suggested that the protein possesses transmembrane(TM) domains and is defined also as a nuclear protein with 69.6%probability by PROST analysis. The potential of the protein totranslocate to the nucleus is supported by the existence of a consensussequence bipartite nuclear localization signal, nuclear functionaldomains, chromo domains, SNF2, Helicase-C and SANT (Table 2). CHROMO(Chromatin Organization Modifier) is a conserved region involved withremodeling of chromatin (FIG. 1B, Table 2). It has been hypothesizedthat the chromo domain may be a vehicle that delivers both positive andnegative transcription regulators to the sites of their action onchromatin. Proteins that contain a chromo domain appear to fall intothree classes. The first class includes proteins having anN-terminal.chromo domain and chromo shadow, mammalian modifier 1 andmodifier 2. The second class includes proteins with single a chromodomain, such as the Drosophilae protein Polycomb (Pc). In the thirdclass, paired tandem chromo domains are found in mammalianDNA-binding/helicase proteins CHD-1 to CHD-4 and yeast protein CHD1.

The existence of two chromodomains followed by DEAD/SNF2 helicase Cdomains suggests that ChroM1 is involved in chromatin regulation.Proteins that contain a N-terminal SNF2 domain appear to be distantlyrelated to the DEAD box helicases however no helicase activity has everbeen demonstrated for these proteins. ChroM1 has similarity to the SNF2subgroup and contains motifs of chromo domain genes; the extendedsequence analysis shows that it belongs to an entirely novel proteinfamily.

Regions with similarity to DEAD-like and helicase DNA binding proteinare well conserved through evolution and are expressed by a series ofviruses and bacteria as transmembrane proteins and in some hypotheticalproteins. This domain is found in proteins involved in a variety ofprocesses including transcription regulation (e.g., SNF2, Brahma), DNArepair (e.g., RAD16, RAD5), and chromatin unwinding (e.g., ISWI).SNF2-domain is recognized also in BRG-1 (human), Brahma BRM (Gallous),kismet (Drosophilae), or SWI/SNF related (Yeast). TABLE 2 Nucleardomains of ChrOM1 Domain Localization Program Chromo domain  688-754;Prosite, SMART 771-829 SNF2  863-1150 Pfam A helicase motif I 880-892helicase motif Ia 912-920 helicase motif III 1023-1030 helicase motif IV1076-1086 helicase motif V 1258-1269 helicase motif VI 1287-1297helicase C domain 1212-1296 Prosite, SMART SANT 1834-1893 SMART, KR-like2429-2436 Bourachot et al, 1999 bipartite NLS 565-585 PSORT NLS1448-1455 1478-1485 1577/9-1584/6 2116-2123 2426-2433 2504-25112537-2542 2632-2637

A SANT-like domain is involved in protein-protein or protein-DNAinteractions. The SANT-like domain following the SNF2 distinguishes theChroM protein from the third class of chromodomain proteins. Thepresence of the SANT domain following Chromo and the presence of theSNF2 and helicase domains conserved through evolution with otherproteins, including the hypothetical protein T04D1.4 from C. elegans,the kismet long isoform of Drosophila and several human hypotheticalproteins. It also appears separately at the Phylogenetic tree based onthe BLASTp algorithm and multiple alignments to construct a new familythat includes ChrOM, AL031667 (human C-Helicase), kismet long isoform(Q9NI64), and T33152 (C. elegans hypothetical protein T04D1.4).

In addition, several domains of unknown function that are shared betweenCHROMO-domain/SNF2 family members were identified, such as CR1-CR3,BRK/TCH and DUF94 (Table 3). TABLE 3 Domains of unknown function inChroM1 protein Domain Localization Program DUF94 1194-1306 SMART CR11492-1559 Therrien et al., 2000 CR2 1616-1706 CR3 1838-1990 TCH/BRK2482-2531 SMART 2556-2600

The ChroM1 protein sequence contains a high percentage (10.5%) of serineamino acid residues which makes the protein acidic. Specifically in exon27, an unusually high number of serine amino acid residues that form a64-amino acid stretch of residues was identified. Database analysisrevealed additional proteins which contain a stretch of serine residuesand are recognized as nuclear phospho-proteins that form multi-proteincomplexes and participate in transcriptional activation (Table 4). Theseproteins are also known to participate in tumorigenic processes.Additionally, the N-terminus of exon 27 contains two conserved domains:a Myc N-terminal domain of an oncoprotein known to be involved in cellreplication and a MAGE domain expressed in a wide variety of tumors(Table 5). TABLE 4 protein with a stretch of serine residues SerineProtein stretch* Function NP_004732 15aa -87% May play an important rolein Nucleolar 132aa -79% transcription catalyzed by RNA phospho-polymerase I protein p130 NP_057417 36aa -89% Splicing co-activator, RNAbinding SRm300 41aa -100% P42568 42aa -100% Transcriptional activatorresulted myeloid/ from chromosomal rearrangement, lymphoidproto-oncogene Or mixed- lineage leukemia NP_002678 61aa -77%Differentiation-specific desmosomal Pinin/Mema/ protein, Nuclearphosphoprotein DRS Pinin was demonstrated in cell membrane andDesmosomes/DRS was demonstrated in the nucleus BAA24570 44aa -84%modulating nucleosome structure and MB20 gene expression during braindevelopment ChrOM1 66aa- 78%*displays percent of serine amino acid in the stretch

TABLE 5 Motifs associated with oncoproteins Domain Description ChrOMSequence Myc-N-term Myc proto-oncogene 2106-2207 MAGE Melanoma antigeneencoding gene 2125-2202

The ChroM1 protein is suggested by the inventors to play a role in theassembly and activity of the nuclear receptors (NR) transcriptioncomplex. The NR box is characterized by a LXXLL sequence flanked with ashort stretch of amino-and carboxyl-terminal amino acids and is bothnecessary and sufficient for ligand-dependent interactions of proteincomplexes with AF2 domains of nuclear receptors. In addition, the BRKmotifs that were identified in other proteins (Table 3) and that wererecognized for their interactions with estrogen or glucocorticoidreceptors, such as BRG1 and BRM, strengthen their function in theregulation of steroid receptors activity.

The ChroM1 protein is believed to be an integral membrane proteinassociated with other proteins (www.softberry.com/protein.html), hasseveral transmembrane domains and contains a GXXXG motif that isimportant in protein interactions. Transmembrane domains and motifs ofinteractions with extracellular or intracellular proteins are summarizedin (Table 6). TABLE 6 Transmembrane domains and motifs of interactionwith extracellular and cytoskeletal proteins Localization Program A.Transmembrane Trans Membrane (TM) 902-923 TMPRED, toppred, TMAP 969-9891252-1276 2454-2474 2640-2660 2682-2702 2797-2820 GxxxG 2693-2697 B.Interaction with Extracellular RGD cell attachment 1544-1546 Prosite LDV2550-2552 LRE 2386-2388 Kringle 2543-2548 Prosite C. Interaction withCytoskeleton Caldesmon 499-670 BLASTp-CD Duplin 512-791 BLASTp(b-catenin interacting protein) Actin 1006-1057 BLOCKs F-actin cappingprotein A subunit 2556-2594 BLOCKs Tropomyosin 1430-1484 BLOCKs2035-2071

The ChroM1 protein is associated with other proteins through cellattachment sites and cytoskeletal components (Table 6). “Blocks” thatsuggest interaction with actin, F actin and thropomyosin are present. Atthe N-terminus, it is suggested that the ChroM1 protein binds tocaldesmon and duplin b-catenin interacting protein. Caldesmon functionsin actin and myosin binding and is implicated in the regulation ofactomyosin interactions that stimulate actin binding of tropomyosin,which binding increases the stabilization of actin filament structure.Caldesmon plays an essential role during cellular mitosis and receptorcapping. Phosphorylation causes caldesmon to dissociate frommicrofilaments and reduces caldesmon binding to actin, myosin, andcalmodulin.

Post-translational modifications: ChroM1 is predicted to be a highlyphosphorylated protein (FIGS. 2A-2C) and it demonstrated phosphorylationof serine and tyrosine residues. It is also a substrate forphosphorylation by protein kinase C (35 sites), protein kinase A (40sites), Casein kinase II (62 sites), Tyrosine kinase (4 sites) andcAMP/cGMP-dependent protein kinase (6 sites), CaMII calmodulin dependentkinase II (26 sites), and p34cdc2 (1 site). Also predicted for multipleO/N glycosylation sites, amidation and myristoylation.

Chromatin remodeling factors participate in transcriptional activationby nuclear receptors. The presence of seven LXXLL nuclear receptoractivation boxes in the ChroM1 sequence (Table 7) suggests that it isbinding to the nuclear receptors. Bioinformatic analysis revealedhomology between ChrOM and members of the SWI/SNF2 complex that wereshown to interact with estrogen and glucocorticoid receptors (Fryer etal., 1998; Ichinose et al., 1997 and Muchardt et al., 1993). The SNF2mammalian homology is BRM and BRG that possess SNF2 and Helicase Cdomain. KR is homologous to HMGI/Y DNA binding domain and was shown tocoordinate the activity of mammalian brm/SNF2alpha (Bourachot et al.,1999). BRK/TCH is a domain of unknown function that is shared betweenSWI/SNF2 and a class of chromodomain proteins and that is included inChrOM1 (Elfring et al., 1998; Daubresse et al., 1999). TABLE 7 Nuclearreceptor (NR) interaction domains Domain Localization Program LxxLL box386-390 SMART 868-872 1036-1040 2031-2035 2659-2663 2721-2725 2793-2797KR-like motif 2429-2436 Bourachot et al. 1999 BRK/TCH 2482-2531 Elfringet al., 1998 2556-2600 Daubresse et al., 1999

The cDNA size was confirmed by Northern blot analysis (data not shown)of RNA extracted from cells representing two model systems of culturedosteoblasts (MSC and TBC; FIG. 3). The RNA was further analyzed toquantify the level of expression of mRNA for ChroM1. MSC and TBC usedfor RNA extraction from each donor was reverse transcribed to cDNA. EachcDNA, obtained from equal amounts of total RNA, was analyzed for ChroMexpression and compared to equal amounts of G3PDH-PCR that served as abaseline for semi-quantitative analysis. Messenger RNA for ChroM1 wasexpressed 2.4-fold higher in MSC than in TBC (p<0.0001, FIG. 3). TheseRNA samples had been previously studied and genomic DNA contaminationwas excluded. MSC is a heterogeneous cell population representing cellsat different stages of differentiation. Thus, the cloning of cells and acomparison for osteogenic potential between cells that possessosteogenic or nonosteogenic capacity was studied when implantedsubcutaneously in mice. ChroM1 expression was 2.5-fold higher inosteogenic than in nonosteogenic clones (FIG. 4, p<0.002).

Antibodies were generated based on the protein sequence and were usedfor protein expression analysis. At the tissue level, paraffin sectionswere analyzed to identify the cells expressing the ChroM1 in bonesections (FIG. 5). Immuno-staining demonstrates that positively stainedosteoprogenitors are located immediately adjacent to the matureosteoblasts (FIG. 5, osteoblast, arrow head). At the bone marrow,unstained cells were counter stained with methylene green.

Freshly isolated bone marrow cells (BMC) were also cultured and anadherent fibroblast-like layer of enriched marrow stromal cell (MSC)population was formed. These two cell populations were analyzed by FACSfor surface antigen expression and the results show that 8%±3ChroM1^(VE+) cells from BMC (FIG. 6) and 55%±18 of MSC from variousdonors were positively stained, which is a 7-fold enrichment ofChroM1^(VE+) cells in the MSC than in BMC (FIG. 6; p>0.0012). Thesecells were also immunostained, and expressed membrane and cytoplasmstaining. Further analysis on the MSC and measurements of cellsestimated the cell size of ChroM1^(VE+) to correspond to >80% ofChroM1^(VE+) cells with a small cells size (less then 30% of maximalcell size) (FIG. 7A). The positive cells were estimated for relativesize and cytoplasm granulation was correlated to small cells having highproliferating capacity (FIG. 7B). ChroM1^(VE+) were correlated withcells that are at G0/G1 as indicated by PI and showed that 76% ofChroM1^(VE+) cells were in the small cell sized fraction. The expressionof ChroM1^(VE+) was correlated with a cell size and a cell cycle stage.In addition, a two-color fluorescence analysis by FACS correlated theChroM1^(VE+) expression with a cell cycle antigen. ChroM1^(VE+) wasassessed by double staining of transcription factors and cyclins thatare markers for cell cycle. A high level for c-Fos, lower levels forc-Jun, Ki-67 and cyclin D1 marked cells at G1 to S phase, and a lowlevel for cyclin B1 that is expressed at S to G2/M stage was detected(FIG. 8). These results summarize the finding that the MSC were doublestained for ChroM1, where the specific markers of cell cycle correlatewith ChroM1^(VE+) cells that are at the G₁ stage and that arecomplementary to the PI staining for DNA content (FIG. 7B).

The ChroM1 expression was correlated with the MSC proliferation capacityin the presence of serum. Cells were cultured and maintained for oneweek in growth medium, and then were replaced with either media forgrowth conditions (10% FCS) or under low. serum (2% FCS) for anadditional week. After the treatment, the cells were released andanalyzed by FACS for ChroM1 expression. Under low serum conditions,lower cell count and a decrease in cell number of ChroM1^(VE+)expression were observed (FIG. 9A). MSC derived from four experiments,demonstrated a significant decrease of 30-45% compared with cells grownunder regular growth conditions (FIG. 9B).

Flow cytometric analyses were used to quantify the co-expression ofChroM1 with a series of other surfaced markers (CD-44, integrins CD-51,CD-61, selections CD-62E, CD-62L, CD-62P and CD-34). In each experiment,the laboratory of the present inventors analyzed four subpopulations(immunonegative cells, ChroM1^(+VE) co-expressing one of the CDmarkers). The analysis was performed with MSC from 5 to 10 differentdonors (FIG. 10 and Table 8). The graph shown in FIG. 10 summarized thelevels of expression for each specific antigen by different donors(shapes) and the respective percent of Chrom1^(+ve) cells that expresseither both antigens (dark bars) or ChroM1 only (white bars). TABLE 8FACS analysis of double staining for MSC Double staining ChroM1 Antigenwith ChroM1* CD only only ChroM1 55 ± 18 (N = 10) CD44 (N = 7) 42 ± 18  52 ± 11 3 ± 4 CD34 (N = 6)  20 ± 8.7 2.25 ± 2  24.5 ± 17   CD51 (N =6) 27 ± 14 3.5 ± 2 18 ± 9  CD61 (N = 6) 33 ± 17    7 ± 4.8 10 ± 6  CD62E(N = 6) 21 ± 12   1 ± 1 19 ± 16 CD62L (N = 6) 19 ± 11 1.2 ± 1 23 ± 17CD62P (N = 6) 18.4 ± 15   2.2 ± 2   24 ± 12.7*Data represent percent of positively stained cells from the total cellpopulation expressed as mean 1 standard deviation. Number of donorschecked appears in brackets.

Functional domains proven for their activity at the N-terminus, an SNF2with ATPase activity (FIG. 11), and at the C-terminus, a KR domain thatfunctions as a DNA binding domain (DBD) (FIG. 12). The protein analysisemployed immunoprecipitates (IP) (FIG. 13) from total cell extracts orbiotinylated preparations of cell membrane, or after cross-linking ofprotein-nucleic acid, with formaldehyde (X-CHIP). This enabled theability to follow the existence and dynamics of protein binding tochromatin. The IP of ChroM1 protein and the protein-DNA complexes weredetected by Western blot and showed a protein of approximately 320 kDa(arrows, FIG. 13). This IP of the protein from total cell extract alsoshowed a lower band of 250 KDa that co-immunoprecipitates with ChroM1(FIG. 13). This band was analyzed by Maldi-mass spectroscopy andelectrospray and identified as a non-skeletal myosin II. Theelectrospray results provide evidence for a complex between chromatinprotein and a cytoskeletal component as was suggested by thebioinformatic analysis. The IP of ChroM1 from MSC extracts was used toquantify ATPase activity and was measured in the presence of stem-loopDNA. ATPase activity determined the Pi as 5 μM release per sample usinga standard curve of inorganic phosphate (FIG. 11). Coupling the ATPasedomain with the DNA binding domain is conserved in the SNF2 familymembers (FIG. 12) and is required for the proper activation of theDNA-dependent ATPase activity. To prove such an interaction, arecombinant protein (rP) that contains the KR region and is recognizedas AT-rich binding site that bind to DNA was used. The KR region isfunctionally and structurally similar to the DNA binding domain ofHMGI/Y proteins. The rP used for EMSA was hybridized with a ³²P-labeledprimer rich in A/T sequence. In the presence of rP and the primer, gelmobility was visualized (FIG. 12B). Two protein concentrations (1 μg and3 μg) were used that result in the increase in binding of theradioactive labeled primer. Addition of a 100-fold excess of the cold(unlabeled) primer abolished the binding of the radiolabeled primer(FIG. 12B). Further analysis to determine if ChroM1 has the availabilityto bind to specific promoters was performed. Using the ChIP assay, threepromoters were identified: estrogen receptor (ERα), BMP4 and osteocalcinthat were amplified by PCR. The PCR amplified from two regions: aproximal promoter (P) to the gene translation initiation and a distalpromoter (D) region that is the 5′ flanking region for the promoter(FIGS. 14, 15, Table 9). The distal region of each promoter was chosenbased on its A/T content, which was 65% to 75%. Estrogen receptor (ER)is highly expressed in proliferating osteoblasts; BMP4 gene from theTGFβ family regulates the first stages of bone matrix mineralization andosteocalcin serves as a marker for osteoblast differentiation. A baselevel for the expression of PCR amplified osteocalcin promoter (SEQ IDNO:34) could be identified but not for BMP4 or ERα promoters. MSC weretreated with 17-β-estradiol (10⁻⁸M) or with TGFβ (5 ng/ml) for 24 hrs.in culture and then were analyzed by the ChIP. A positive signal for theERα promoter following estrogen treatment and for BMP4 promoterfollowing TGFβ treatment was detected (FIG. 14). The quality of DNA wasanalyzed also in the corresponding input chromatin fraction. This PCRfragment from the distal region was used further as a probe for EMSAwith the recombinant protein (rp). Each labeled probe was analyzed whenbound to the recombinant protein with a shift of the complex, the gelshift result with detection of higher band (FIG. 15). The binding wasreduced in the presence of distamycin A, an antibiotic that blocks thebinding to A/T rich regions. Distamycin A is an antibiotic,characterized by an oligopeptidic pyrrolocarbamoyl frame ending with anamidino moiety, which binds reversibly to the DNA minor groove with highselectivity for A/T-rich sequences and shows antiviral andanti-protozoal activity (Cozzi P, Mongelli N., 1998) TABLE 9 Size of PCRPromoter Primers product BMP4 BMP dF GCTAAAGGAGCACAATGCCT (SEQ ID NO:20)145 bp BMP dR CCCCAAAAGGAGGACAAAAT (SEQ ID NO:21) BMP4 BMP pFTAGTACCTCCGCACGTGGTC (SEQ ID NO:22) 457 bp BMP pR CTGCAGGCTCGAGATAGCTT(SEQ ID NO:23) Osteocalcin OC dF ACCAGCCTACAGGCTCTTTTT (SEQ ID NO:24)113 bp OC dR AGAGCCAGACCCTGTCTCAA (SEQ ID NO:25) Osteocalcin OC pFAGGCTGCCTTTGGTGACTC (SEQ ID NO:26) 497 bp OC pR TTATACCCTCTGGGCTGTGC(SEQ ID NO:27) Estrogen Erα dF CGCATGATATACTTCACCTATTTTT (SEQ ID NO:28)204 bp receptor α Erα dR TTGGGCTAGGATATGCAGAA (SEQ ID NO:29) EstrogenErα pF AACAGCCTCCTGTCTACCGA (SEQ ID NO:30) 110 bp receptor α Erα pRCAGGAGAAAGGAGCATGGAC (SEQ ID NO:31)Expression of ChroM in Embryogenesis

The presence of the ChroM1 protein was identified also during embryonicdevelopment. Immunostaining of sections from mouse embryo at 16.5 daysresulted with staining in mesenchymal condensation during skeletaldevelopment and its appearance with development (FIG. 16). Positivestaining was also detected in progenitors at the skeletal muscle (FIG.17). These results suggest that the identification of progenitors giverise to two cell lineages (osteogenic and skeletal). The histologicalstaining detects the mesenchymal progenitors on the developmental level.

Histopathology of Human Bone Tumors

The ChroM1 expression in tissue sections of normal bone (FIG. 18A) orcultured MSC (FIG. 18B) was membranous or cytoplasmic. Immunostaining ofbiopsies of bone cancer osteogenic sarcoma (OS), the most frequentprimary, highly malignant tumor cells, occur in poorly differentiatedcells. Such cells produce the malignant state and appear mainly inchildren or young adults and are shown in FIG. 18C. The tumor cells ofosteosarcoma were examined in biopsies and the cells lines were observedto express a different cellular pattern of the protein, where theprotein is translocated to the nucleus (FIG. 18D).

The cDNA of other alternative splice forms were cloned (FIGS. 19 and20A-20D) and further computer predicted splice forms were then alsoverified by RT-PCR and the predicted transcripts were obtained (Table10). TABLE 10 Identified and predicted splice variants of ChroM Splicevariant Translation initiation Unique feature Protein domainsGENE/PROTEIN-SCHEME ChrOM1 + 5′ of exon 1 ChrOM2 + 1) missing 5′ and 3′of 1 and SANT, KR-like translation of 36 bp only 2) missing exon 27ChrOM3 + 5′ of exon 35: 5′ UTR+ translation TM Hypothetical protein + 5′UTR of exon 7: SNF2 FLJ12178 3′ UTR of exon 12 EST AND PARTIAL CDNAVERIFIED BY RT-PCR * ChrOM4 * Extra 3 bp 5′ exon 28 Kiaa0308 + Missing3′ exon 29 BE882430 (ChroM5) + Extra exon 1a Hook, b-catenine bindingW88543 * Extra exon 26a AAA TRIP 13 like Spleen/fetal liver ChroM *Extra exon 15p Signal peptide Pred1 ChroM * Extra exon 23p Signalpeptide Pred21Not verified by RT-PCR from MSC W99387 from fetal heart,2Not expressed in RT-PCR of MSC AA009999 fetal spleen

Having now fully described this invention, it will be appreciated bythose skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

While this invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications. This application is intended to cover any variations,uses, or adaptations of the inventions following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth as follows in the scope of theappended claims.

All references cited herein, including journal articles or abstracts,published or corresponding U.S. or foreign patent applications, issuedU.S. or foreign patents, or any other references, are entirelyincorporated by reference herein, including all data, tables, figures,and text presented in the cited references. Additionally, the entirecontents of the references cited within the references cited herein arealso entirely incorporated by references.

Reference to known method steps, conventional methods steps, knownmethods or conventional methods is not in any way an admission that anyaspect, description or embodiment of the present invention is disclosed,taught or suggested in the relevant art.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art (including the contents of thereferences cited herein), readily modify and/or adapt for variousapplications such specific embodiments, without undue experimentation,without departing from the general concept of the present invention.Therefore, such adaptations and modifications are intended to be withinthe meaning and range of equivalents of the disclosed embodiments, basedon the teaching and guidance presented herein. It is to be understoodthat the phraseology or terminology herein is for the purpose ofdescription and not of limitation, such that the terminology orphraseology of the present specification is to be interpreted by theskilled artisan in light of the teachings and guidance presented herein,in combination with the knowledge of one of ordinary skill in the art.

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1. An isolated polypeptide having the activity of a chromatin remodelingprotein, comprising: (a) the amino acid sequence of SEQ ID NO:2; (b) afragment of (a); (c) an amino acid sequence having at least 95% sequenceidentity to the amino acid sequence of SEQ ID NO:2; or (d) a fragment of(c), wherein said fragment of (a) or of (c) has the activity of thechromatin remodeling protein of SEQ ID NO:2.
 2. The isolated polypeptideof claim 1, which comprises the amino acid sequence of SEQ ID NO:2. 3.An isolated naturally occurring variant of the polypeptide of claim 2,comprising the amino acid sequence of SEQ ID NO:4.
 4. A moleculecomprising the antigen binding portion of an antibody against thevariant polypeptide of claim
 3. 5. An isolated naturally occurringvariant of the polypeptide of claim 2, comprising the amino acidsequence of SEQ ID NO:6.
 6. A molecule comprising the antigen bindingportion of an antibody against the variant polypeptide of claim
 5. 7. Anisolated naturally occurring variant of the polypeptide of claim 2,comprising the amino acid sequence of SEQ ID NO:8.
 8. A moleculecomprising the antigen binding portion of an antibody against thevariant polypeptide of claim
 7. 9. The isolated polypeptide of claim 1,which comprises a fragment of (a).
 10. The isolated polypeptide of claim1, which comprises an amino acid sequence having at least 95% sequenceidentity to the amino acid sequence of SEQ ID NO:2.
 11. The isolatedpolypeptide of claim 1, which comprises a fragment of (c).
 12. Acomposition, comprising the polypeptide of claim 1 and an excipient,diluent, carrier or auxiliary agent.
 13. A molecule comprising theantigen binding portion of an antibody against the polypeptide ofclaim
 1. 14. The molecule of claim 13, which is a polyclonal antibody.15. The molecule of claim 13, which is a monoclonal antibody.
 16. Amethod for recovering human stromal progenitor cells from a cell mixturederived from human bone marrow, comprising: contacting the cell mixturewith a molecule of claim 13 which is an antibody that selectively bindsto an antigen on human osteogenic and muscle progenitor cells; andrecovering antibody-bound cells from the cell mixture.
 17. The method ofclaim 16, wherein the antigen on human osteogenic progenitor cells whichis selectively bound with the molecule is a polypeptide comprising theamino acid sequence of SEQ ID NO:2.
 18. The method of claim 16, whereinthe human stromal progenitor cells are isolated culture-expanded humanosteogenic progenitor cells.
 19. A method for separating a cellpopulation containing human osteogenic progenitor cells from a cellmixture derived from human bone marrow, comprising: contacting the cellmixture with a molecule of claim 13 which is an antibody thatselectively binds to an antigen on human osteogenic progenitor cells;and separating antibody-bound cells from the cell mixture.
 20. Themethod of claim 19, wherein the antigen on human osteogenic progenitorcells which is selectively bound with the molecule is a polypeptidecomprising the amino acid sequence of SEQ ID NO:2.
 21. The method ofclaim 19 wherein the antibody-bound cells are labeled with a fluorescenttag and the fluorescent tagged, antibody-bound cells are separated byfluorescence activated cell sorting.
 22. The method of claim 19, whereinthe antibody is a polyclonal antibody which is immobilized on a solidsupport prior to the step of contacting.
 23. The method of claim 22,wherein the solid support is a column or magnetic beads.
 24. The methodof claim 19, wherein the human osteogenic progenitor cells are isolated,culture-expanded human osteogenic progenitor cells.
 25. The method ofclaim 19, further comprising: contacting the separated antibody-boundcells with a second antibody which selectively binds to a second antigenon a subpopulation of human osteogenic progenitor cells; and separatingthe second antibody-bound cells as a subpopulation from the populationof human osteogenic progenitor cells.
 26. The method of claim 25,wherein the subpopulation of second antibody-bound cells is an isolated,culture-expanded subpopulation of human osteogenic progenitor cells. 27.An isolated population of cells enriched for human osteogenic progenitorcells, wherein greater than 30% of said population of cells are positivefor the presence of the polypeptide of SEQ ID NO:2 and can differentiateinto osteogenic cells and muscle cells.
 28. The isolated population ofcells of claim 27, wherein greater than 40% of said population of cellsare positive for the presence of the polypeptide of SEQ ID NO:2 and candifferentiate into osteogenic cells and muscle cells.
 29. The isolatedpopulation of cells of claim 27, wherein greater than 50% of saidpopulation of cells are positive for the presence of the polypeptide ofSEQ ID NO:2 and can differentiate into osteogenic cells and musclecells.
 30. The isolated population of cells of claim 27, wherein greaterthan 60% of said population of cells are positive for the presence ofthe polypeptide of SEQ ID NO:2 and can differentiate into osteogeniccells and muscle cells.
 31. A composition comprising a physiologicallyacceptable medium and the isolated population of cells of claim
 27. 32.A method of treating bone or muscle tissue damage, comprisingadministering to a patient in need thereof the isolated population ofcells enriched for human osteogenic progenitor cells of claim 27 whichare culture expanded and which are autologous to the patient.
 33. Amethod of generating bone or muscle tissue, comprising: seeding a matrixor scaffold for generating bone or muscle tissue with the isolatedpopulation of cells enriched for human osteogenic progenitor cells ofclaim 27 that have been culture expanded, inducing differentiation ofthe culture expanded population of cells to generate bone or muscletissue using the matrix or scaffold as support.
 34. A method foridentifying osteosarcoma cells in a tissue sample, comprising:contacting a tissue sample containing osteogenic progenitor cells with amolecule of claim 13; and detecting by immunohistochemistry the presenceof the molecule in the nuclei of cells from tissue biopsies to identifyosteosarcoma cells.
 35. A method for evaluating the effectiveness of atreatment for osteogenic progenitor sarcoma, comprising: contacting atissue sample containing osteogenic progenitor cells with a molecule ofclaim 13; and detecting by the immunohistochemistry the presence of themolecule in nuclei of cells in the tissue sample to identifyosteosarcoma progenitor cells and to evaluate the effectiveness of thetreatment.
 36. A method of screening for and identifying an enhancer orinhibitor compound that affect expression of the chromatin remodelingprotein ChroM1, comprising: incubating a human cell, which expresses theChroM1 polypeptide of SEQ ID NO:2, in the presence or absence of apotential enhancer or inhibitor compound that affects expression ofChroM1; determining the level of expression of the ChroM1 polypeptide inthe presence of the potential enhancer or inhibitor compound relative tothe level of expression of the ChroM1 polypeptide in the absence of thepotential enhancer or inhibitor; and identifying as an enhancer compoundany potential enhancer or inhibitor compound for which said determiningstep determines that the level of expression of the ChroM1 polypeptidein the presence of the potential enhancer compound is substantially morethan that in the absence of the potential enhancer compound andidentifying as an inhibitor compound any potential enhancer or inhibitorcompound for which said determining step determines that the level ofexpression of ChroM1 polypeptide in the presence of the potentialinhibitor compound is substantially less than that in the absence of thepotential inhibitor compound.
 37. The method of claim 36, furthercomprising a step of isolating the enhancer or inhibitor identified insaid identifying step.
 38. A method for treating osteoporosis,comprising administering an enhancer compound identified in the methodof claim 36 to a patient in need thereof.
 39. A method for treatingosteopetrosis or osteosarcoma, comprising administering an inhibitorcompound identified in the method of claim 36 to a patient in needthereof.
 40. A method of identifying A/T-rich promoter regions of genesinvolved in modulation of osteoblast differentiation and capable ofbinding to the DNA binding domain of the ChroM1 chromatin remodelingprotein, comprising: contacting fragments of genomic DNA with a peptidecontaining a DNA binding domain comprising the amino acid sequence ofresidues 2429-2437 of SEQ ID NO:2; identifying a fragment of genomic DNAbound by said peptide; determining the nucleotide sequence of thepromoter region on said fragment bound by said peptide to identify anA/T-rich promoter region of a gene involved in modulation of osteoblastdifferentiation.
 41. The method of claim 40, wherein said peptidecontaining a DNA binding domain comprises the amino acid sequence ofresidues 2333 to 2480 of SEQ ID NO:2.
 42. A method of screening for andidentifying a compound which stimulate differentiation of osteogenicprogenitor cells, comprising: incubating human cells, which express theChroM1 chromatin remodeling protein of SEQ ID NO:2, with a potentialstimulator compound; adding formaldehyde to the incubated human cells tocrosslink proteins to DNA in chromatin by in vivo fixation; sonicatingthe crosslinked chromatin to solubilize the crosslinked chromatin;immunoprecipitating the solubilized crosslinked chromatin withantibodies specific for the ChroM1 protein of SEQ ID NO:2 to formimmunocomplexes; recovering DNA from the immunocomplexes; incubatingunder amplification conditions the recovered DNA with oligonucleotideprimers capable of amplifying an A/T-rich promoter region of a geneinvolved in control of osteoblast cell differentiation if present in therecovered DNA; and detecting an amplification product and identifying asa stimulator compound any potential stimulator compound for which anamplification product corresponding to said AT-rich promoter region isdetected step.
 43. The method of claim 42, wherein the A/T-rich promoterregion is selected from the group consisting of the human estrogenreceptor (ER) promoter and the human bone morphogenic protein (BMP)promoter.
 44. The method of claim 43, wherein the human estrogenreceptor promoter is the human estrogen receptor α (ERα) promoter. 45.The method of claim 43, wherein the human bone morphogenic proteinpromoter is the human bone morphogenic protein-4 (BMP-4) promoter. 46.An isolated nucleic acid molecule, comprising a nucleotide sequenceencoding the polypeptide of claim
 1. 47. The isolated nucleic acidmolecule of claim 46, wherein the polypeptide comprises the amino acidsequence of SEQ ID NO:2.
 48. The isolated nucleic acid molecule of claim46, wherein said nucleotide sequence encoding said polypeptide comprisesnucleotides 88 to 8781 of SEQ ID NO:1.
 49. The isolated nucleic acidmolecule of claim 46, wherein the polypeptide comprises a fragment of(a).
 50. The isolated nucleic acid molecule of claim 46, wherein thepolypeptide comprises an amino acid sequence having at least 95%sequence identity to the amino acid sequence of SEQ ID NO:2.
 51. Theisolated nucleic acid molecule of claim 46, wherein the polypeptidecomprises a fragment of (c).
 52. A vector, comprising the nucleic acidof claim
 46. 53. A host cell transformed with the nucleic acid of claim46.
 54. An isolated nucleic acid molecule which hybridizes to thenucleotide sequence of the nucleic acid molecule of claim 46 under highstringency conditions.
 55. A process for preparing a polypeptidecomprising the amino acid sequence of SEQ ID NO:2 or a fragment thereof,comprising: recombinantly expressing the polypeptide from the nucleicacid molecule of claim 46; and recovering the expressed polypeptide toprepare the polypeptide comprising the amino acid sequence of SEQ IDNO:2 or a fragment thereof.
 56. An antisense oligonucleotidecomplementary to a messenger RNA comprising nucleotides 88 to 8781 ofSEQ ID NO:1 and encoding a polypeptide having the activity of achromatin remodeling protein, wherein said oligonucleotide inhibits theproduction of said polypeptide.
 57. A method for treating osteopetrosisor osteosarcoma, comprising administering the antisense oligonucleotideof claim 56 to a patient in need thereof.
 58. An isolated nucleic acidmolecule, comprising a nucleotide sequence encoding the naturallyoccurring variant polypeptide of claim
 3. 59. The isolated nucleic acidmolecule of claim 58, wherein said nucleotide sequence comprisesnucleotides 148 to 3558 of SEQ ID NO:3.
 60. An isolated nucleic acidmolecule, comprising a nucleotide sequence encoding the naturallyoccurring variant polypeptide of claim
 5. 61. The isolated nucleic acidmolecule of claim 60, wherein said nucleotide sequence comprisesnucleotides 402 to 1298 of SEQ ID NO:5.
 62. An isolated nucleic acidmolecule, comprising a nucleotide sequence encoding the naturallyoccurring variant polypeptide of claim
 7. 63. The isolated nucleic acidmolecule of claim 62, wherein said nucleotide sequence comprisesnucleotides 196 to 1206 of SEQ ID NO:7.
 64. An isolated DNA molecule,comprising a nucleotide sequence encoding the amino acid sequence of SEQID NO:2, wherein said nucleotide sequence consists of the sequence of afragment of human genomic DNA.